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以改良CTAB法提取越桔(Vaccinium Linn.)品种薄雾DNA为材料,采用正交设计L16(45)表探讨了Tag酶;Mg~(2+);DNA;dntps及引物浓度5个主要因素对越桔ISSR-PCR扩增反应的影响,通过直观分析和方差分析结合,建立了越桔ISSR-PCR(20μL)最佳反应体系,各组分的浓度为:Tag酶0.75 U,Mg~(2+)1.875 mmol/L,DNA 0.8mmol/L,dntp 0.1mmol/L,引物0.2μmol/L.在此基础上探讨了引物UBC835的最佳退火温度、循环次数和延伸时间,结果分别为54.2℃、35次、60s.选用两个引物对21份越桔资源进行ISSR扩增,结果显示优化的体系稳定性较高.
Five main factors of Tagging enzyme, Mg ~ (2 +); DNA; dntps and primer concentration were investigated by orthogonal design of L16 (45) table by using improved CTAB method to extract DNA of Vaccinium Linn. The optimal reaction system of ISSR-PCR (20μL) for bilberry was established by visual analysis and analysis of variance (ANOVA). The concentration of each component was: Tag enzyme 0.75 U, Mg ~ ( 2+) 1.875 mmol / L, DNA 0.8 mmol / L, dntp 0.1 mmol / L and primer 0.2 μmol / L. The optimum annealing temperature, number of cycles and extension time of primer UBC835 were discussed. The results were 54.2 ℃, 35 times, 60s. Two primers were used to amplify 21 ISSRs of bilberry resources. The results showed that the optimized system had higher stability.